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Detailed knowledge of biological variation can facilitate accurate interpretation of clinical pathology parameters. A recent biological variation study in Asian elephants (Elephas maximus) found that hematology parameters had high individuality, which suggests that population-derived reference intervals may be an insensitive diagnostic tool. In elephant medicine, sensitive hematology-related diagnostics are crucial for clinical decision-making, particularly in elephants at risk for elephant endotheliotropic herpesvirus hemorrhagic disease (EEHV-HD). The objective of this study was to assess biological variation of hematology parameters in African elephants to determine whether population-derived reference intervals are a sensitive diagnostic tool for interpreting results and to provide a useful alternative. Eight healthy African elephants had blood collected under behavioral training every other week for 8 wk. Complete blood cell count (CBC) analysis was performed in duplicate to assess analytical variation. Previous methods were used to determine between-individual variation, within-individual variation, index of individuality, and reference change values (RCV). This study found that most hematology parameters displayed intermediate-to-high individuality, which suggests that alternatives to population-derived reference intervals are necessary to detect pathologic changes. To test the results of our biological variation data, a case of EEHV-HD was retrospectively evaluated. Individual normal values and calculated RCV detected a clinically significant monocytopenia, leukopenia, and thrombocytopenia associated with EEHV2 viremia. However, none of these parameters fell outside a population-derived reference interval. This study highlights the utility of biological variation in clinical decision-making and demonstrates that individual normal values and RCV may be important diagnostic tools for CBC interpretation in African elephants.

The diagnostic workup of veterinary patients is a decision-making process. For the decision-making processes to be consistent, established decision criteria are essential. These criteria may be in the form of decision limits or population-based and subject-based reference intervals (RIs). One solution to the frequent problem of small reference sample sizes is to harvest results from laboratory databases, a procedure called data mining. The suitability of a particular population for RI generation assumes homogeneity in that population. Unlike RIs that are descriptive of the distribution of blood values in healthy subjects, a decision limit is a predetermined test threshold that distinguishes between two subgroups of a target population. Reference intervals typically are printed on laboratory reports along with patient results. This only should be done when the provided RIs are applicable to the patient demographic.

INTRODUCTION: Monitoring of clinical effectiveness of bypassing agents in haemophilia patients is hampered by the lack of validated laboratory assays. Thromboelastography (TEG) and rotational thromboelastometry (ROTEM) have been evaluated for predicting clinical effectiveness of bypassing agents, however, with limited success. AIM: Application of a longitudinal model-based approach may allow for a quantitative characterization of the link between ROTEM parameters and the probability of bleeding events. METHODS: We analyse longitudinal data from haemophilia A rats receiving gene-based FVIIa prophylaxis in terms of total circulatory levels of FVII/FVIIa, clotting time (CT) measured using ROTEM and the probability of bleeding events. RESULTS: Using population pharmacokinetic-pharmacodynamic (PKPD) modelling, a PK-CT-repeated time-to-event (RTTE) model was developed composed of three submodels (a) a FVII/FVIIa PK model, (b) a PK-CT model describing the relationship between predicted FVIIa expression and CT and (c) a RTTE model describing the probability of bleeding events as a function of CT. The developed PK-CT-RTTE model accurately described the vector dose-dependent plasma concentration-time profile of total FVII/FVIIa and the exposure-response relationship between AAV-derived FVIIa expression and CT. Importantly, the developed model accurately described the occurrence of bleeding events over time in a quantitative manner, revealing a linear relationship between predicted change from baseline CT and the probability of bleeding events. CONCLUSION: Using PK-CT-RTTE modelling, we demonstrated that ROTEM parameters can accurately predict the probability of bleeding events in a translational animal model of haemophilia A.

Introduction: Monitoring of clinical effectiveness of bypassing agents in haemophilia patients is hampered by the lack of validated laboratory assays. Thromboelastography (TEG) and rotational thromboelastometry (ROTEM) have been evaluated for predicting clinical effectiveness of bypassing agents, however, with limited success. Aim: Application of a longitudinal model-based approach may allow for a quantitative characterization of the link between ROTEM parameters and the probability of bleeding events. Methods: We analyse longitudinal data from haemophilia A rats receiving gene-based FVIIa prophylaxis in terms of total circulatory levels of FVII/FVIIa, clotting time (CT) measured using ROTEM and the probability of bleeding events. Results: Using population pharmacokinetic-pharmacodynamic (PKPD) modelling, a PK-CT-repeated time-to-event (RTTE) model was developed composed of three submodels (a) a FVII/FVIIa PK model, (b) a PK-CT model describing the relationship between predicted FVIIa expression and CT and (c) a RTTE model describing the probability of bleeding events as a function of CT. The developed PK-CT-RTTE model accurately described the vector dose-dependent plasma concentration-time profile of total FVII/FVIIa and the exposure-response relationship between AAV-derived FVIIa expression and CT. Importantly, the developed model accurately described the occurrence of bleeding events over time in a quantitative manner, revealing a linear relationship between predicted change from baseline CT and the probability of bleeding events. Conclusion: Using PK-CT-RTTE modelling, we demonstrated that ROTEM parameters can accurately predict the probability of bleeding events in a translational animal model of haemophilia A.

Background: Hemophilic arthropathy is a debilitating morbidity of hemophilia caused by recurrent joint bleeds. We investigated if the joint bleed volume, before initiation of treatment, was linked to the subsequent degree of histopathological changes and the development of bone pathology in a mouse model of hemophilic arthropathy.Methods: FVIII knock-out (F8-KO) mice were dosed with a micro-CT blood pool agent prior to induction of hemarthrosis. Eight hours after induction, the bleed volume was quantified with micro computed tomography (micro-CT) and recombinant FVIII treatment initiated. On Day 8, inflammation in the knees was characterized by fluorescence molecular tomography. On Day 14, knee pathology was characterized by micro-CT and histopathology. In a second study, contrast agent was injected into the knee of wild-type (WT) mice, followed by histopathological evaluation on Day 14.Results: The average joint bleed volume before treatment was 3.9 mm3. The inflammation-related fluorescent intensities in the injured knees were significantly increased on Day 8. The injured knees had significantly increased synovitis scores, vessel counts, and areas of hemosiderin compared to un-injured knees. However, no cartilage- or bone pathology was observed. The bleed volume before initiation of treatment correlated with the degree of synovitis and was associated with high fluorescent intensity on Day 8. In F8-KO and WT mice, persistence of contrast agent in the joint elicited morphological changes.Conclusion: When applying a delayed on-demand treatment regimen to hemophilic mice subjected to an induced knee hemarthrosis, the degree of histopathological changes on Day 14 reflected the bleed volume prior to initiation of treatment.

Population-based reference intervals (RIs) are vital tools used to characterize health and disease based on laboratory values. The science and statistical basis for RI generation have evolved over the past 50 yr. Current veterinary-specific guidelines by the American Society of Veterinary Clinical Pathology exist for establishing RIs from nondomestic and wild animals. A list of 35 items that should be included during generation and publication of reference data was distilled from the currently available RI guidelines. The archives of five peer-reviewed journals were searched and 106 articles presenting laboratory reference data from nondomestic or wildlife species were identified and each reviewed by two authors to determine compliance with the list of 35 items. A compliance score was calculated as the number of articles that fulfilled the item out of the number where it would have been appropriate to fulfill the item. Most articles reported the number of reference individuals (compliance score 0.98), their partitioning demographics (compliance score 0.95), and sample collection and handling practices (compliance scores 0.97 and 0.96, respectively). Common deficiencies included omitting discussion of the validation status of the analytical methods for the species being evaluated (compliance score 0.12), documentation of use of exclusion criteria (compliance score 0.51), outlier detection (compliance score 0.43), appropriate statistical methods for the reference population (compliance score 0.34), and calculation and presentation of confidence intervals around the reference limits (compliance score 0.35). Compliance scores were not statistically different when stratified on the number of individuals in the largest and smallest evaluated group or the format of the article (full vs short format). Articles that cited RI generation guidelines fulfilled more of the required steps and provided a more complete description of their data (compliance score 0.74) than those

Introduction: Haemophilic animal models are used to study blood-induced cartilage damage, but quantitative and sensitive outcome measures are needed. Aim: To develop a novel quantitative method for detecting early cartilage degeneration in a haemophilic rat model of blood-induced joint damage. Methods: The 35Sulphate incorporation (35SO4 2− assay) was applied to tibial and patellar cartilage of wild-type rats to quantify baseline proteoglycan synthesis and to evaluate the effect of 4-day blood exposure in vitro. Next, haemarthrosis was induced in 39 FVIII-deficient rats and characterized by changes in knee joint diameter and development of bone pathology (using micro-CT). Four- and 16-day posthaemarthrosis proteoglycan synthesis rate (PSR) was assessed using the 35SO4 2− assay, with the contralateral knee as control. Results: In vitro, a decrease in PSR in tibial and patellar cartilage was demonstrated following blood exposure. In vivo, joint diameter and development of bone pathology confirmed successful induction of haemarthrosis. In the blood-exposed knee, tibial and patellar PSR was inhibited 4 and 16 days after induced haemarthrosis. Interestingly, at day 16 the proteoglycan synthesis in the contralateral knee was also inhibited to an extent correlating with that of the blood-exposed knee. Conclusion: For the first time, early changes in cartilage matrix synthesis upon blood exposure were quantified with the 35SO4 2− assay in a haemophilic rat model, establishing this assay as a novel method to study blood-induced cartilage damage.

The aim of this study was to objectively evaluate the biological variation of healthy Asian elephant (Elephas maximus) hematology and biochemistry parameters, therefore enabling evidence-based clinical decision-making to improve patient management. Ten clinically healthy elephants had blood samples collected weekly for 5 wk under standardized conditions. The analytical, between- and within-individual variation, index of individuality, and reference change values were calculated using previously reported methods. Large between-individual variation and small within-individual variation for almost all parameters indicated that individual normal values should be used for interpreting blood results from Asian elephants.